Vol.

176,

May

15, 1991

No.

BIOCHEMICAL

3, 1991

AND

RESEARCH

BIOPHYSICAL

COMMUNICATIONS Pages

1062-1067

MOLECULAR CLONINGOF cDNA ENCODING THE 16 KDa SUBUNITOF VACUOLARHt-ATPase FROMMOUSECEREBELLUM Hironori

Hanada, Masahisa Hasebe, Yoshinori Moriyama, Masatomo Maeda, and Masamitsu Futai

Department of Organic Chemistry and Biochemistry, Institute of Scientific and Industrial Research, Osaka University, 8-l Mihogaoka, Ibaraki; Osaka 567, Japan Received April

1, 1991

cDNA for the 16 kDa subunit of vacuolar Ht-ATPase was cloned from mouse cerebellum and sequenced. The deduced polypeptide (155 amino acid residues; molecular weight, 15,808) was highly hydrophobic and homologous to the subunits of bovine adrenal medulla, Torpedo marmorata electric lobe, Drosophila and yeast. Glu-139 (supposed to be essential for proton transport) was also conserved as the potential dicyclohexylcarbodiimide binding site. The subunit had four transmembrane segments: Segment II and IV were highly homologous and Glu-139 was located in Segment IV. The roles of the non-conserved regions are e 1991Academic Press,Inc. discussed.

Vacuolar H+-ATPase (V-ATPase) has been found in endomembranesystems of eukaryotic gradients

cells

and shown to translocate

that are essential

for organellar

hormones and neurotransmitters Its function hydrolases similar

in acidifying (2).

structure

functions

such as accumulation of

granules (for review,

V-ATPase is unable to synthesize

to FoF1 ATPase (ATP synthase) and consists The peripheral

sectors.

hydrolysis

and a similar

complicated

sector

has a catalytic

subunit

structure

membranesector consists of at least 2 different

and 20 kDa, and acts as a proton pathway. to the membrane sector

Abbreviations

and inhibits

1062

1).

for lysosomal ATP, it

has a

of membraneand site(s)

to that

for

of F1.

ATP The

subunits of 16 kDa

The 16 kDa subunit

used: V-ATPase, vacuolar H’-ATPase; DCCD, dicyclohexylcarbodiimide.

0 1991 by Academic Press, Inc. in any form reserved.

of reproduction

see ref.

Baf ilomycin A1 specifically

ATPase (3).

0006-291X/91$1.50 Copyright All rights

to form electrochemical

lysosomes provides optimal conditions

Although

peripheral

intrinsic

in secretory

protons

binds of V-

Vol.

176,

No.

3, 1991

BIOCHEMICAL

ATPase and the c subunit a common ancestral cation

AND

of F,Fl

protein

RESEARCH

ATPase have been suggested

(4),

of the 16 kDa subunit

BIOPHYSICAL

COMMUNICATIONS

to be derived

and as in the case of the c subunit, with

dicyclohexylcarbodiimide

from

modifi-

(DCCD) inhibits

ATPase activity. Recently,

Moriyama

constituent

and Futai

of the synaptic

membrane protein).

granules,

of neurotransmitters.

ATPases of these other

vesicles

organelles

different

of this

problem,

which

to focus

study,

mouse cerebellum.

roles

to those

marmorata

electric

lobe

homologies

to

latter

the

is

(8)) two

pump for

to the V-ATPases and lysosomes,

in different

Drosophila

(9),

subunits

were

of

this

adrenal

and the role

found

in

or whether For

protein

on

in

the

V-ATPase

(6,

7).

the 16 kDa subunit

of

subunit

showed

medulla

(4),

and yeast lower

V-

subunit

of the entire for

the

organelles.

intrinsic

a cDNA coding

from bovine

accu-

to know whether

a major

sequence

of the subunit

properties

seems to be a suitable

in assembly

acid

of the subunits

The topology

it

and sequenced

The amino

homology

granules

a major

20 % of the total

as a primary

interest

are expressed

because

is

the same enzymatic

similar

the 16 kDa subunit

attention,

we cloned

of

as chromaffin

membrane and has essential In this

is

V-ATPase

about

and functions It

of V-ATPase

that

(constituting

are structurally

such

isoforms

study

formers.

vesicles

reported

This ATPase has essentially

as V-ATPase of chromaffin mulation

(5)

than

(lo), those

Torpedo

although to

of non-conserved

high

the

its two

regions

are discussed. MATERIALS AND METHODS A mouse (ICR) cerebellum cDNA library constructed with bacteriophage Phage plaques were transkgtll was propagated in E. coli Y1090 (r-e&) (11). ferred to nitrocellulose filters (Schleicher and Schiill) and hybridized with an equimolar mixture of four 65-base synthetic oligonucleotides corresponding to the antisense (positions 367-431 and 478542) and sense (positions 422-486 and 533-597) strands of the carboxyl terminal half of the bovine 16 kDa subunit (4). Ten positive clones were isolated from 250,000 plaques. Although they were classified into four groups (I, hmvpl, 3, 5, 128; II, ;imvp8, 20, maps, portions of their se163; III, ~mvp4; VI, Amvp2) from the restriction The cDNAs encoding the amino terminal region quences overlapped (Fig. 1). were amplified from the library by the polymerase chain reaction with Taq DNA polymerase (Perkin Elmer Cetus) using 24-base primers corresponding to the 1gtll left arm (GGTGGCGACGACTCCTGGAGCCCG) and antisense strand (position 328The cDNAs were subcloned into pBluescript II SK M13(+) and both 341, Fig. 1). strands were sequenced (12). 1063

Vol.

176,

No.

BIOCHEMICAL

3, 1991

AND

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RESEARCH

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Restriction enzymes, T4 DNA ligase, T4 polynucleotide kinase and the fragment of E. coli DNA polymerase I were purchased from Takara Shuzo (Osaka, Jap3az) and Nippon Gene (Toyama, Japan). (Kyoto, Japan), Toyobo PlATP (5,000 Ci/mmol) were purchased [,-32P)dCTP (3,000 Ci/mmol)and [7from AmershamCorp. Sequenase was from United State Biochem. Corp. A random priming DNA labeling kit was from Boeringer-Mannheim. All other chemicals used were the highest grade commercially available. large

RESULTSANDDISCUSSION The nucleotide subunit

of V-ATPase

fragments amplified tional

initiation

sequence of the cDNA encoding the was determined

using

hybridization-positive

by the polymerase chain reaction site

was assigned to the Met-l

sequences around the initiation

found between the in-frame termination (Fig.

2).

subunit

A termination

was found next

the

polyA

tail

(Fig

signal

1).

(4) and its

codon (13).

and

The transla-

similarity

to

No Met codon was

-135) and Met-l

the reading

to the Lys-155 codon.

(557 bp), there was a potential

(Fig.

codon (position

codon (TAG) for

clones

codon from the homology of

the sequence to that of the bovine adrenal subunit the eukaryotic

cerebellum 16 kDa

mouse

codon

frame of the 16 kDa

In the 3’-noncoding

for polyadenylation

region

(14) upstream of

2).

Pairwise comparisons of the nucleotide

sequences of the

16 kDa subunit with those of bovine adrenal medulla (4),

mouse

cerebellum

the Torpedo marmorata

100 bp mvp2

f >

-

1

Molecular cloning of cDNA encoding the 16 KDa subunit of vacuolar H(+)-ATPase from mouse cerebellum.

cDNA for the 16 kDa subunit of vacuolar H(+)-ATPase was cloned from mouse cerebellum and sequenced. The deduced polypeptide (155 amino acid residues; ...
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